US5286638A - Peroxidase gene of microbial origin - Google Patents

Peroxidase gene of microbial origin Download PDF

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US5286638A
US5286638A US07/792,259 US79225991A US5286638A US 5286638 A US5286638 A US 5286638A US 79225991 A US79225991 A US 79225991A US 5286638 A US5286638 A US 5286638A
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peroxidase
amino acid
seq
protein
peptide
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Yoshikazu Tanaka
Toshihiko Ashikari
Haruyo Hatanaka
Yuji Shibano
Teruo Amachi
Toru Nakayama
Motoo Sumida
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Suntory Holdings Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)

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  • This invention relates to a cDNA encoding a peroxidase originating from a microorganism (Arthromyces ramosus) and a process for producing the peroxidase by use of host cells containing said gene.
  • a microorganism Arthromyces ramosus
  • peroxidases which are enzymes capable of oxidizing various compounds in the presence of hydrogen peroxide, have been used in the same way as various other oxidases as a clinical diagnostic reagent in assays of glucose, cholesterol, phospholipids, urea and so forth. These enzymes have also been used as a labelled enzyme in enzyme linked immunoassays. They have mainly been produced from plants such as horseradish and Japanese radish. However peroxidases originating from these plants contain isozymes having slightly differing properties from each other, and which therefore require considerable labour costs in order to purify them to such a degree that they are usable as a diagnostic reagent.
  • peroxidases of microbial origin have been known.
  • cytochrome c peroxidases and NADH peroxidases produced by bacteria or fungi are unsuitable as a clinical or diagnostic reagent from the viewpoint of the specificity thereof, since they are generally less specific than the common ones originating from horseradish or Japanese radish.
  • peroxidases capable of acting on o-dianisidine as a hydrogen donor have been produced from Eschelichia coli or microorganisms belonging to the genus Myrothecium.
  • this enzyme is also unsuitable for the aforesaid diagnostic use due to the carcinogenic nature of o-dianisidine.
  • the present inventors conducted research to obtain a naturally occurring peroxidase of microbial origin, which is usable as, for example, a clinical diagnostic reagent or a labelled enzyme in enzyme linked immunoassays similar to conventional ones originating from horseradish or Japanese radish.
  • the inventors have already reported a peroxidase produced by a fungus belonging to the genus Arthromyces, which peroxidase has the desired properties (Japanese Patent Laid-Open No. 43987/1986).
  • peroxidase of the genus Arthromyces is far superior to conventional peroxidases in terms of chemiluminescence generating activity in the case of determining with chemiluminescent reagent, for example, peroxides, as a clinical diagnostic reagent or a labelled enzyme in an enzyme linked immunoassay (refer to Japanese Patent Laid-Open No. 219398/1988).
  • the Arthromyces peroxidase is an ideal enzyme to be used as a clinical diagnostic reagent or in an enzyme linked immunoassay as described above, the problem was that this peroxidase cannot be produced at a reasonable cost. This is because a large scale culture of a fungus such as Arthromyces, is very difficult.
  • genetic engineering would be the most suitable approach.
  • the gene encoding the aforesaid peroxidase of Arthromyces origin hitherto has not been obtained. Thus it was impossible to produce said enzyme on a large scale or to modify the same by use of protein engineering techniques.
  • FIG. 1 illustrates the steps to the formation of an intermediate plasmid pYE2006 in the construction of plasmid pYEPOD1 which expresses the peroxidase protein in yeast.
  • FIG. 2 illustrates the steps from pYE2006 to pYE22m.
  • FIG. 3 illustrates the steps from pYE22m to the final pYEPOD1 plasmid.
  • FIG. 4 illustrates electrophoretic patterns showing that the yeast transformed with pYEPOD1 produced the peroxidase protein.
  • FIGS. 5(a), (b) and (c) illustrate the nucleotide sequence of the cDNA of Arthromyces peroxidase including portions of the 3' and 5' non-coding regions (see SEQ ID NO:16). The amino acid sequence encoded by the cDNA is also given.
  • the present inventors have conducted extensive studies in order to solve the above problems. As a result, they have successfully obtained a cDNA of Arthromyces peroxidase and clarified the nucleotide sequence of said gene and the amino acid sequence of said enzyme, thus completing the present invention.
  • an appropriate host such as E. coli or a yeast which is easy to grow and further to modify said peroxidase by genetic engineering techniques.
  • the present invention provides a peroxidase gene of Arthromyces origin or a variant or a mutant thereof having a substantially identical biological activity, a recombinant vector containing said gene, host cells transformed with such a plasmid containing said gene and a process for producing the peroxidase by growing said transformants.
  • the peroxidase gene of the present invention may be obtained from a fungus belonging to the genus Arthromyces.
  • Arthromyces ramosus which was named SAM 003 and deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology under the accession number of FERM BP-838, is available therefor.
  • substantially identical used herein is intended to mean that, with regard to the characteristics of the peroxidase encoded by the gene of the present invention, any enzyme which has substantially the same biological activity as the enzyme obtained by the process described in Japanese Patent Laid-Open No. 43987/1986 and has the preferable characteristics described in Japanese Patent Laid-Open No. 219398/1988 is encompassed herein.
  • Isolation of the mRNA from the aforesaid starting microorganism, preparation of a cDNA library and screening of said library may be performed by a known method, e.g., the one described in Molecular Cloning, second ed., (by Sambrook et al., Cold Spring Harbor, 1988).
  • the gene encoding the peroxidase of the present invention can be obtained in the following manner.
  • polyA RNAs are extracted from Arthromyces cells.
  • cDNAs can be prepared to be inserted into a phage cloning vector (for example, ⁇ gt10) which is then used to transform a host such as E. coli.
  • the resulting cDNA library is screened by use of a synthetic DNA probe corresponding to the partial amino acid sequence of the peroxidase.
  • positive clones containing a DNA fragment encoding the target peroxidase are selected.
  • the DNA probe to be used in the screening can be synthesized by purifying the peroxidase protein from a culture of Arthromyces by the method as described hereinafter.
  • the amino acid sequences of at least some portions thereof are determined, whereby an appropriate probe can be chosen based on said sequences.
  • a longer probe fragment can be prepared by the polymerase chain reaction (PCR) techniques.
  • a longer cDNA may be prepared by the following procedure. Namely, the phage DNA is prepared from one of the positive clones and an appropriate EcoRI fragment from the phage DNA can be used to screen the same library. Thus several positive clones will be obtained. These positive phase clones are digested with EcoRI and the separated DNA fragments are subcloned into an appropriate vector (for example, M13mp18 or M13mp19). The nucleotide sequences of the thus inserted DNA fragments are determined by, for example, the dideoxy sequencing method.
  • the purified peroxidase protein are analyzed to determine the amino acid composition and the amino acid sequences in the amino and carboxyl terminal regions and the results are compared with the amino acid sequence deduced from the nucleotide sequence of the DNA as determined by the aforesaid method.
  • Examples of the host cells for expressing the cDNA include bacteria such as E. coli and Bacillus subtilis, yeasts and fungi.
  • the peroxidase of the present invention can be produced by host cells which have been transformed with a plasmid containing the peroxidase gene, preferably in combination with the signal sequence for said gene, together with an appropriate promoter and a terminator.
  • the aforesaid transformant is cultured in a medium containing a suitable carbon source, nitrogen source and trace metal elements in accordance with the method of Shinmen et al. (Agric. Biol. Chem., 50, 247-249, 1986).
  • the target enzyme is purified from the cell extract or, preferably, from the culture supernatant by a combination of known purification procedures such as precipitation, absorption, filtration through a molecular sieve and electrophoresis.
  • the cell extract or the culture supernatant is subjected to ammonium sulfate precipitation (at approximately 75% saturation) followed by a combination of column chromatography steps (e.g., DEAE-cellulose column chromatography and Ultrogel AcA44 column chromatography).
  • ammonium sulfate precipitation at approximately 75% saturation
  • column chromatography steps e.g., DEAE-cellulose column chromatography and Ultrogel AcA44 column chromatography.
  • the amino acid composition of the peroxidase protein was analyzed by use of a commercial peroxidase of Arthromyces origin (available from Suntory, Ltd.). First, the amino acid composition was analyzed by a conventional method described in detail in Seikagaku Jikken Koza 1, Tanpakushitsu no Kagaku II (by Takahashi, Ikenaka, et al., Tokyo Kagaku Dojin) and Zoku Seikagaku Jikken Koza 2, Tanpakushitsu no Kagaku (Vol. 1) (by Tsunazawa, Sakiyama, et al.). First, 5 nmole of the peroxidase protein was sealed in a glass tube together with 6N hydrochloric acid and hydrolyzed at 110° C. for 24 hours. Then the free PTH-amino acids contained in the reaction mixture were quantitated in an amino acid analyzer (Hitachi Automatic Amino Acid Analyzer 835) to determine the amino acid composition (refer to Table 1).
  • the protein was carboxymethylated under reducing conditions, followed by purifying by reverse phase HPLC. Namely, 100 mg (2.5 ⁇ mole) of the peroxidase was dissolved in 3.0 ml of a denaturation buffer solution [Tris.HCl (pH 8.5) containing 6M Gdn.HCl, 10 mM EDTA.2 Na) and incubated at 50° C. for 1 hour. Next, 143 ⁇ mole (10 ⁇ l) of 2-mercaptoethanol was added thereto and the atmosphere of the reaction system was replaced by nitrogen. After an incubation at 37° C.
  • the yield of the protein was 16%.
  • SDS-PAGE only two protein molecular species (molecular weight: approximately 40 K and 30 K) were detected from the protein fraction.
  • the amino acid sequence of the N-terminal region of of the carboxymethylated peroxidase protein obtained above was analyzed with a gas phase protein sequencer (Shimadzu Seisakusho, Ltd.). As a result, no free PTH-amino acid was detected, which indicated that the N-terminal amino acid was protected. Thus the N-terminus of the carboxymethylated peroxidase protein was deprotected in the following manner.
  • the protected residue at the N-terminal was revealed to be pyroglutamyl and further the amino acid sequence of the first 20 residues in the N-terminal region was determined as follows. [This corresponds to positions 21 (Gln) to 40 (Asn) in FIG. 5]:
  • (Gln) represents a pyroglutamic acid residue (see SEQ ID NO:1)
  • the carboxymethylated peroxidase (48 nmol, 1.9 mg) was suspended in 100 ⁇ l of 0.1M NH 4 HCO 3 (pH 7.9), to which 2 ⁇ l of a 1% solution of TPCK-Trypsin (Worthington) in 0.0024N HCl was added and the mixture was incubated at 35° C. for 6 hours. White clouding in the liquid disappeared immediately after the addition of the enzyme and the solution became clear. After the completion of the reaction, the mixture was lyophilized to stop the reaction. The dry residue was dissolved in 70% formic acid to be provided to the next step of HPLC.
  • the peptide fragments of the trypsin digest were separated by reverse phase HPLC under the following conditions:
  • peptide 1 positions from 125 (Ala) to 154 (Arg); peptide 2: positions from 162 (Ser) to 186 (Arg); peptide 3: positions from 240 (Gly) to 261 (Phe); peptide 4: positions from 275 (Thr) to 288 (Val); peptide 5: positions from 300 (Met) to 307 (Arg); and peptide 6: positions from 325 (Ala) to 336 (Asp).
  • RNA fraction was prepared by the method using guanidine thiocyanate/caesium chloride. Further, polyA.RNA fraction was separated therefrom by use of oligo(dT)cellulose.
  • oligo(dT)cellulose The details of the guanidine thiocyanate/caesium chloride method and the purification of polyA.RNA by oligo(dT)cellulose can be found in, for example, R. McGookin, Robert J. Slater et al. (Methods in Molecular Biology, vol. 2, Humana Press Inc., 1984).
  • RNAs were then fractionated in an oligo(dT)cellulose column. Namely, a column was packed with oligo(dT)cellulose and equilibrated with 1 ⁇ column binding solution [20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.5M NaCl, 0.2% SDS]. Next, the RNA precipitate was dissolved in a column eluting solution [20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.2% SDS] and incubated at 65° C. for 5 minutes. An equal volume of 2 ⁇ column binding solution was added and the solution was passed through the oligo (dT) cellulose column which had been equilibrated.
  • RNA fraction was eluted and recovered.
  • 2M sodium acetate was added at the final concentration of 0.15M. After being allowed to stand at -80° C. overnight, the mixture was centrifuged. The precipitate thus formed was washed twice with 70% ethanol, dried under a reduced pressure and dissolved in sterilized water.
  • the polyA.RNAs thus obtained were used as the templates to prepare cDNAs with a commercial cDNA synthesizing kit "cDNA Synthesis System Plus” (Amersham, Co.) in accordance with the recommendation by the manufacturer.
  • the resulting cDNAs were inserted into the E. coli phage vector ⁇ gt10 and the vector was then introduced into an E. coli strain such as C600HF1 (available from Clone Tech, Co.) to thereby give a cDNA library.
  • an E. coli strain such as C600HF1 (available from Clone Tech, Co.) to thereby give a cDNA library.
  • a commercial kit "cDNA cloning system ⁇ gt10" was used in accordance with the recommendations by the manufacturer.
  • the synthetic DNA fragments used in this example had nucleotide sequences presumed based on a partial amino acid sequence of the peroxidase and they were synthesized with a DNA Synthesizer 371A (Applied Bio-System, Co.).
  • a PCR reaction was performed as follows.
  • the complementary chains were synthesized from the polyA.RNAs purified above by a cDNA synthesis kit.
  • the resulting cDNA were amplified by the PCR reaction with use of a set of synthetic DNA fragments 5'-CCCTGCAGGATCCATGTGGCA(AG)TC(GATC)ATGAC-3', (see SEQ ID NO:8) comprising a linker region and a nucleotide sequence corresponding to the partial amino acid sequence Trp-Gln-Ser-Met-Thr, and (see SEQ ID NO:9) one other synthetic DNA fragment 5'-GCGAGCTCGGTACCCGGGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
  • the reaction mixture was prepared by using GeneAmp TMKit (Takara Shuzo Co., Ltd.) in accordance with the instructions given in the kit.
  • a cycle comprising reaction at 94° C. for 1.5 minutes, at 45° C. for 2.5 minutes and at 72° C. for 3.4 minutes was repeated 25 times.
  • the amplified cDNA were cleaved with KpnI and BamHI and cloned into plasmid M13mp18 and M13mp19.
  • the restriction sites were those present in the primers existing at each end.
  • a clone containing a nucleotide sequence corresponding to the partial amino acid sequence of the peroxidase in addition to the primer sequences was identified.
  • a partial fragment of the peroxidase gene could be obtained.
  • a DNA fragment (approximately 0.4 kb) containing the partial sequence of the peroxidase gene was used as a probe, and a cDNA library of approximately 5,000 clones was screened in the following manner.
  • the ⁇ gt10 cDNA library was plated in such a manner as to give approximately 1,000 plaques per plate and the plates were incubated at 37° C. Each plate was covered with a nylon membrane (Amersham, Co.) which was pinholed at several points with an injection needle so as to memorize the relative locations of the membrane and the plate. Next, the membrane was removed and it was layered with the plaques upside on a filter paper which had been impregnated with a denaturation solution (1.5M NaCl, 0.5M NaOH).
  • a denaturation solution 1.5M NaCl, 0.5M NaOH
  • the membrane After being allowed to stand as such for 7 minutes, the membrane was layered on a filter paper impregnated with a neutralization solution (1.5M NaCl, 0.5M Tris-HCl, pH 7.2, 0.001M EDTA) and allowed to stand for 5 minutes. After air-drying, the membrane was placed on an UV trans-illuminator with the plaque side downward and irradiated for 2 to 5 minutes to thereby fix the DNA.
  • a neutralization solution 1.5M NaCl, 0.5M Tris-HCl, pH 7.2, 0.001M EDTA
  • the DNA-DNA hybridization was performed in accordance with the method of Jeffrey and Flavell (Cell 12: 439-439, 1977). Namely, the membrane filter, on which the DNA had been fixed, was immersed in a hybridization solution (6 ⁇ SSC, 5 ⁇ Denhard's solution, 0.5% SDS, 10 ⁇ g/ml salmon sperm DNA) at 65° C. for 30 minutes. Then the membrane was placed into a thick nylon bag and the probe DNA labeled with 32 P (10 6 to 10 8 cpm/ ⁇ g) was added thereto. After a reaction in the hybridization solution at 65° C.
  • a hybridization solution 6 ⁇ SSC, 5 ⁇ Denhard's solution, 0.5% SDS, 10 ⁇ g/ml salmon sperm DNA
  • the membrane filter was washed in a washing buffer solution [5 ⁇ SSC, 0.1% SDS (W/V)] at 65° C. for 15 minutes four times.
  • the membrane filter was then dried and subjected to autoradiography at -80° C. with X-ray film and a sensitized paper sheet.
  • Phage DNA was prepared from each positive clone obtained above in accordance with the instructions attached to the ⁇ gt10 kit. After being digested with EcoRI, the phage DNA was subjected to agarose gel electrophoresis. After the completion of the electrophoresis, the appropriate fragment was excised from the gel and the DNA was collected and purified by using Gene Clean (Bio 101, Co.) in accordance with the instructions given therein. After being extracted with phenol/chloroform and precipitated with ethanol, the DNA was ligated with E. coli phage vector M13mp18, which had been digested with EcoRI and dephosphorylated with an alkaline phosphatase, and used to transform E. coli strain JM109.
  • the encoding region corresponded to an amino acid sequence consisting of 364 residues and ranged from the initiator codon ATG to the terminator codon TGA given in FIG. 5.
  • the amino acid sequence in the amino terminal region of the peroxidase as determined in Example 1 initiated with Gln at position 21 in FIG. 5 and the 20th amino acid residues after said Gln (i.e., up to Asn at position 40) completely agreed with the one deduced from the cDNA sequence.
  • the full-length cDNA was excised from the clone C13 containing the full-length cDNA of Arthromyces peroxidase obtained in Example 2 and inserted into a yeast expression plasmid. Thus a peroxidase expressing plasmid in yeast was constructed.
  • the plasmid thus obtained was referred to as pYEPOD1 (FIG. 3).
  • POD should be expressed in this plasmid under the control of glycelaldehyde 3-phosphate dehydrogenase promoter.
  • the transformant G-1315 (pYEPODl) was incubated in 5 ml of Burkholder's medium [P. R. Burkholder, Am. J. Bot., 30, 206 (1943) ] containing 1% casamino acids under shaking at 30° C. for 48 hours. 1 ml of the culture broth was collected and the supernatant was concentrated approximately 50-fold by Ultra-Free C3GC (Milipore Co.).
  • the yeast cells were treated by the method of Yaffe et al. [Proc. Natl. Acad. Sci. USA, 81, 4819 (1984) ] to obtain proteins.
  • the enzyme activity was determined by the following method. To 1 ml of a 0.1M potassium phosphate buffer solution (pH 7.0), 1.3 ml of a 11.5 mM phenol solution, 0.25 ml of a 10 mM 4-aminoantipyrine solution and 0.2 ml of a 6 mM hydrogen peroxide solution were added. After pre-heating the mixture at 37° C., 0.25 ml of the sample was added and the mixture was reacted for 5 minutes. After the completion of the reaction, 0.2 ml of a 20% sodium azide solution was added and the absorbance at 500 nm was measured. The obtained value was referred to as a reaction value.
  • a control value was determined by adding 0.2 ml of a 20% sodium azide solution before adding the sample and then the reaction was performed.
  • the absorbance measured in the same manner was referred to as the control value.
  • the titer of the peroxidase was expressed in Unit (U), namely, the amount of the enzyme capable of consuming 1 mol of hydrogen peroxide within 1 minute was referred to as 1 U.
  • the titer (U/ml) of the sample peroxidase was calculated in accordance with the following equation.
  • ⁇ A 500 represents the difference between a reaction value and a control value.
  • the extracellular supernatant showed an activity 15.6 mu/ml whereas the crude cell extract showed no activity.
  • the protein concentration in the extracellular supernatant was 0.53 mg/ml and the specific activity of POD was 290 u/ml. Based on these data, it was estimated that approximately 0.01% of the extracellular proteins represented the active POD protein.
  • the transformant WA802 (pKPOD1) was incubated in 2 ml of LB medium (AP 50 ⁇ g/ml, IPTG 1 mM) under shaking at 37° C. for 15 hours. Cells were harvested from 1 ml of the culture and a cellular protein fraction was prepared by the method of Yaffe et al. Proteins, reacting with anti-Arthromyces peroxidase antibody were then detected by the aforesaid enzyme-labelled antibody method. As a result, a single band was observed at the same position as that of the Arthromyces peroxidase protein. Since plasmid-free host WA802 did not exhibit such a band, it was considered that the Arthromyces peroxidase protein was produced in the recombinant E. coli.
  • the cDNA of a peroxidase gene of microbial origin has been provided and the nucleotide and amino acid sequences thereof have been clarified.
  • a yeast and E. coli transformed with a plasmid containing the aforesaid cDNA produce the protein identical with the Arthromyces peroxidase.
  • the enzyme activity was detected in the case of the yeast.
  • the present invention has enabled the production of the peroxidase of Arthromyces origin on a large scale by use of genetic engineering techniques and further to modify the peroxidase molecule through protein engineering techniques.

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5851811A (en) * 1992-06-01 1998-12-22 Novo Nordisk A/S Peroxidase variants with improved hydrogen peroxide stability
US20070154993A1 (en) * 2004-09-15 2007-07-05 Olson John S Enhancing Recombinant Hemoglobin Production By Co-Expression With Alpha Hemoglobin Stabilizing Protein
US20070166792A1 (en) * 2004-09-15 2007-07-19 Olson John S Increasing hemoglobin and other heme protein production in bacteria by co-expression of heme transport genes
US20070172924A1 (en) * 2004-09-15 2007-07-26 Olson John S Increasing the stability of recombinant adult human apohemoglobin

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0730641A1 (de) * 1993-10-26 1996-09-11 Novo Nordisk A/S Myxokokkae oxidase
NL9401048A (nl) * 1994-03-31 1995-11-01 Stichting Scheikundig Onderzoe Haloperoxidasen.
CN1301739A (zh) * 1999-12-24 2001-07-04 上海博德基因开发有限公司 一种新的多肽——过氧化物酶蛋白11和编码这种多肽的多核苷酸
WO2023225459A2 (en) 2022-05-14 2023-11-23 Novozymes A/S Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0171074A2 (de) * 1984-08-07 1986-02-12 Suntory Limited Peroxidase und Verfahren zu deren Herstellung
US5137822A (en) * 1987-08-19 1992-08-11 Director-General Of Agency Of Industrial Science & Technology An Organ Of The Ministry Of Industrial Trade And Industry Of Japan Transformed yeasts having steroid-hydroxylase activity and process for hydroxylation of steroids using the said transformed yeasts
WO1992016634A1 (en) * 1991-03-22 1992-10-01 Novo Nordisk A/S A process for producing heme proteins

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0171074A2 (de) * 1984-08-07 1986-02-12 Suntory Limited Peroxidase und Verfahren zu deren Herstellung
US5137822A (en) * 1987-08-19 1992-08-11 Director-General Of Agency Of Industrial Science & Technology An Organ Of The Ministry Of Industrial Trade And Industry Of Japan Transformed yeasts having steroid-hydroxylase activity and process for hydroxylation of steroids using the said transformed yeasts
WO1992016634A1 (en) * 1991-03-22 1992-10-01 Novo Nordisk A/S A process for producing heme proteins

Non-Patent Citations (14)

* Cited by examiner, † Cited by third party
Title
Belyavsky, A. et al. "PCR-based cDNA library construction . . . " Nuc. Acids Res. 17(8) 2919-2932 (Apr. 1989).
Belyavsky, A. et al. PCR based cDNA library construction . . . Nuc. Acids Res. 17(8) 2919 2932 (Apr. 1989). *
Berger, S. L. et al. "Guide to Molecular Cloning Techniques" (1987) Meth. in Enzynol. 152:393-399, 415-423, 432-447, 661-704.
Berger, S. L. et al. Guide to Molecular Cloning Techniques (1987) Meth. in Enzynol. 152:393 399, 415 423, 432 447, 661 704. *
Deutscher, M. P. "Guide to Protein Purification" Meth. in Enzynol. 182:602-613, 738-751 (1990).
Deutscher, M. P. Guide to Protein Purification Meth. in Enzynol. 182:602 613, 738 751 (1990). *
Fishel, Laurence A., et al., "Yeast Cytochrome c Peroxidase; Mutagenesis and Expression in Escherichia Coli Show Tryptophan-51 is Not the Radical Site in Compound I", Biochemestry, vol. 26, (1987) pp. 351-360.
Fishel, Laurence A., et al., Yeast Cytochrome c Peroxidase; Mutagenesis and Expression in Escherichia Coli Show Tryptophan 51 is Not the Radical Site in Compound I , Biochemestry, vol. 26, (1987) pp. 351 360. *
Ortlepp, S. A. et al. "Expression and Characterization of a . . . " J. Biotechnol. 11:353-364 (1989).
Ortlepp, S. A. et al. Expression and Characterization of a . . . J. Biotechnol. 11:353 364 (1989). *
Saloheimo, Markku et al. "A Lingin Peroxidase-Encoding cDNA from the White-Rot Fungus Phlebia Radiata; Charaterization and Expression in Trichoderma Reesei", Gene 85 (1989) pp. 343-351.
Saloheimo, Markku et al. A Lingin Peroxidase Encoding cDNA from the White Rot Fungus Phlebia Radiata; Charaterization and Expression in Trichoderma Reesei , Gene 85 (1989) pp. 343 351. *
Saloheimo, Markku et al., "Heterologous Production of a Ligninolytic Enzyme; Expression of the Phlebia Radiata Laccase Gene in Trichoderma Reesei", Biotechnology, vol. 9, (1991) pp. 987-990.
Saloheimo, Markku et al., Heterologous Production of a Ligninolytic Enzyme; Expression of the Phlebia Radiata Laccase Gene in Trichoderma Reesei , Biotechnology, vol. 9, (1991) pp. 987 990. *

Cited By (7)

* Cited by examiner, † Cited by third party
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US5851811A (en) * 1992-06-01 1998-12-22 Novo Nordisk A/S Peroxidase variants with improved hydrogen peroxide stability
US6258769B1 (en) * 1992-06-01 2001-07-10 Novozymes A/S Patents Peroxidase variants with improved hydrogen peroxide stability
US20070154993A1 (en) * 2004-09-15 2007-07-05 Olson John S Enhancing Recombinant Hemoglobin Production By Co-Expression With Alpha Hemoglobin Stabilizing Protein
US20070166792A1 (en) * 2004-09-15 2007-07-19 Olson John S Increasing hemoglobin and other heme protein production in bacteria by co-expression of heme transport genes
US20070172924A1 (en) * 2004-09-15 2007-07-26 Olson John S Increasing the stability of recombinant adult human apohemoglobin
US7642233B2 (en) 2004-09-15 2010-01-05 William Marsh Rice University Enhancing recombinant hemoglobin production by co-expression with alpha hemoglobin stabilizing protein
US7803912B2 (en) 2004-09-15 2010-09-28 William Marsh Rice University Increasing the stability of recombinant adult human apohemoglobin

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EP0486067A3 (en) 1993-04-07
ES2085403T3 (es) 1996-06-01
EP0486067B1 (de) 1996-01-31
ATE133709T1 (de) 1996-02-15
JPH04228078A (ja) 1992-08-18
CA2055698A1 (en) 1992-05-17
DK0486067T3 (da) 1996-02-26
GR3018850T3 (en) 1996-05-31
EP0486067A2 (de) 1992-05-20
CA2055698C (en) 2002-03-26

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